PROJECT SUMMARY Hospital-acquired secondary infections are an escalating problem of global significance. Indeed, multi-drug resistant Pseudomonas aeruginosa is the leading cause of hospital-acquired infections in the USA and P. aeruginosa is now a priority pathogen on the CDC ESKAPE pathogen list. P. aeruginosa infection is a particular problem in cystic fibrosis, microbial keratitis, in third-degree burn units, and in cancer sufferers and HIV patients. P. aeruginosa virulence and biofilm development depend on the bacterial cell-to-cell communication process called quorum sensing. The known P. aeruginosa quorum-sensing circuit possesses two canonical LuxI/R type signaling pathways: LasI/R and RhlI/R, that, together, control an estimated 10% of the genes in the genome. The known circuit functions as follows: LasI produces and LasR responds to the autoinducer 3OC12-homoserine lactone. The LasR:3OC12-homoserine lactone complex activates transcription of many genes including rhlR, encoding a second quorum-sensing receptor. RhlR binds to the autoinducer C4-homoserine lactone, the product of RhlI. RhlR:C4-homoserine lactone also directs a large regulon of genes including those encoding virulence factors such as pyocyanin, elastases, rhamnolipids and genes required for biofilm formation. Typically, mutations in quorum-sensing luxI-type and luxR-type genes (i.e., lasI-lasR and rhlI-rhlR) confer identical phenotypes because each component of the pair needs the other to function. However, using biofilm analyses, transcriptional reporter assays, RNA-seq studies, and animal infection assays, I discovered that RhlR directs both RhlI- dependent and RhlI-independent regulons. Importantly, I showed that ?rhlI mutant cell-free culture fluids, i.e., that lack C4-homoserine lactone, contain an activity that stimulates RhlR-dependent gene expression. I hypothesize that RhlR responds to an alternative ligand, in addition to the traditional C4-homoserine lactone autoinducer. Supporting this notion, I showed that the enzyme PqsE is required for alternative ligand synthesis. Finally, I demonstrated that while the RhlR-RhlI system is dispensable, the RhlR-PqsE system is the crucial quorum-sensing system required for biofilm formation and for virulence in two animal models of infection. Here, I propose to 1) determine the chemical structure of the alternative ligand and define how it interacts with RhlR; 2) characterize the PqsE active site, discover small molecule inhibitors of PqsE, and identify and characterize additional factors involved in alternative ligand synthesis; 3) map the alternative ligand and/or C4-homoserine lactone-dependent RhlR regulon(s) required for biofilm development at the single cell level and at the level of community. The proposed research will contribute significant insights about the chemical lexicon used by a clinically important bacterium in biofilms and in disease. Moreover, the proposed research will provide a mechanistic understanding of how quorum sensing regulates virulence and biofilm formation, which is crucial for understanding basic P. aeruginosa biology and for successful development of anti-quorum-sensing strategies.